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Swine streptococcosis is a significant threat to the Chinese pig industry, and Streptococcus equi ssp. zooepidemicus (SEZ) is one of the major pathogens. SEZ ATCC35246 is a classical virulent strain, while SEZ ST171 is a Chinese attenuated vaccine strain. In this study, we employed stable isotope labeling by amino acids in cell culture and liquid chromatography-mass spectrometry (LC-MS) to determine the differential response of macrophages to infection by these two strains. Eighty-seven upregulated proteins and 135 downregulated proteins were identified. The proteomic results were verified by real-time polymerase chain reaction for 10 chosen genes and Western blotting for three proteins. All differentially abundant proteins were analyzed for their Gene Ontology and Kyoto Encyclopedia of Genes and Genomes annotations. Certain downregulated proteins were associated with immunity functions, and the upregulated proteins were related to cytomembrane and cytoskeleton regulation. The phagocytosis rate and cytokine genes transcription in Raw264.7 cells during SEZ ATCC35246 and ST171 infection were detected to confirm the bioinformatics results. These results showed that different effects on macrophage phagocytosis and cytokine expression might explain the different phenotypes of SEZ ATCC35246 and ST171 infection. This research provided clues to the mechanisms of host immunity responses to SEZ ST171and SEZ ATCC35246, which could identify potential therapy and vaccine development targets.

Streptococcus equi ssp. zooepidemicus (SEZ) causes meningitis in both humans and animals. Some dissociative proteins of SEZ are cytotoxic to mouse brain microvascular endothelial cells (mBMECs) and may contribute to the penetration of SEZ across the blood-brain barrier (BBB). In this study, the ability of SEZ to penetrate across an in vitro BBB model was confirmed. We used stable isotope labeling with amino acids in cell culture (SILAC) to label SEZ proteins with heavy or light isotope-tagged amino acids, along with LC-MS/MS to determine which SEZ proteins were involved in interactions with mBMECs. The efficiency of SEZ protein isotope labeling was 94.7 %, which was sufficient for further analysis. Forty-nine labeled peptides were identified as binding to mBMECs, which matched to 25 SEZ proteins. Bioinformatic analysis indicated that most of these proteins were cytoplasmic. These proteins may have functions in breaching the host BBB, and some of them are known virulence factors in other bacteria. Indirect immunofluorescence results indicated that SEZ enolase had binding activity toward mBMECs. Protective test results showed that enolase was a protective antigen against SEZ infection. This research is the first application of SILAC combined with LC-MS/MS to identify SEZ proteins that may contribute to the infection of mBMECs and potentially show functions related to breaching the BBB. The outcomes provide many future avenues for research into the mechanism of SEZ-induced meningitis.

Phages are abundant and ubiquitous in nature,and are therefore important components of microbial communities.The investigation on the survival environment of bacteriophage in the nature might lay a foundation for the further study on regulating function of phage in the microecological environment.To understand the ecology of mycobacteriophage,we collected different types of soil samples from three areas in China to determine the isolation rates of phages and analyzed the relationship between isolation rates and four affecting factors in this study.The mycobacteriophages were isolated from 50 soil samples with mixed lyosol of citric acid and disodium hydrogen phosphate dodecahydrate as extractant.The isolation result was detected by the double agar method and dipping method.At the same time,pH value,moisture,organic carbon level and cation exchange capacity of samples were measured.Thirteen strains of mycobacteriophages were isolated and named from CJAUS6 to CJAUS18.The morphological observation under the transmission electron microscope showed that,all the isolates have a regular icosohedron head [(86.5±5.83—108±23.2)nm ×(83.2±3.04—93.2±5.92)nm ] and a contract tail [(73.0±8.36—87.7±1.86)nm ×(19.3±1.44—30.7±8.78)nm].So they can be classified as a member of the order Caudovirales and the family Myoviridae.The isolation results from five types of soils indicated that the isolation rate(41.2%) from dark brown earth was highest,followed by yellow brown earth(25.0%) and drab soil(16.7%),while no phages were isolated from the heilu soil and brown earth samples.The reason might relate to three factors as follows: firstly,the organic carbon level is the most in the dark brown earth;secondly,the influence of weathering and eluviatin on dark brown soil is weaker than that on yellow brown earth and drab soil;thirdly,the dark brown earth in this paper was affected by microclimate.In order to know the relationship between the isolation rate and affecting factors,the two-dimentional coordinates graphs were drawn by means of Microsoft Office Excel Software.The result showed that the isolation rate was the highest when the pH value was between 7.45 and 7.95,and moisture was between 13.7% and 21.7%,and the curve of their relationship was expressed as a same inverted "V" form.When the cation exchange capacity was between 20.8cmol/kg and 28.6cmol/kg,the isolation rate had a positive proportion with cation exchange capacity.It is supposed that the way in which cation exchange capacity affects isolation rate might attribute to the change of concentration and composition of anions and cations in the soil solution.And we found the concentration and composition of anions and cations is proportional to the adsorption quantity of phages.There was no obvious correlation between the isolating rate and organic carbon capacity.The results indicated that the soils types and physical and chemical properties of soils might contribute to the isolation rates of mycobacteriophage.Further work will be performed on the relationships between bacteriophage pI and soil pH values,and between water content in soil and the titres of host bacteria.

Streptococcus suis is a swine pathogen and also a zoonotic agent. The formation of biofilms allows S. suis to become persistent colonizers and resist clearance by the host immune system and antibiotics. In this study, biofilm forming potentials of various S. suis strains were characterized by confocal laser scanning microscopy (CLSM), scanning electron microscopy (SEM) and tissue culture plates stained with crystal violet. In addition, the effects of five antimicrobial agents on biofilm formation were assayed in this study. S. suis produced biofilms on smooth and rough surface. The nutritional contents including glucose and NaCl in the growth medium modulated biofilm formation. There was a significant difference in their biofilm-forming ability among all 46 S. suis strains. The biofilm-forming potential of S. suis serotype 9 was stronger than type 2 and all other types. However, biofilm formation was inhibited by five commonly used antimicrobial agents, penicillin, erythromycin, azithromycin, ciprofloxacin, and ofloxacin at subinhibitory concentrations, among which inhibition of ciprofloxacin and ofloxacin was stronger than that of other three antimicrobial agents.Our study provides a detailed analysis of biofilm formation potential in S. suis, which is a step towards understanding its role in pathogenesis, and eventually lead to a better understanding of how to eradicate S. suis growing as biofilms with antibiotic therapy.

The highly conserved DNAs of Equine herpesvirus-1 (EHV-1), Equine arteritis virus (EAV), Equine influenza virus (EIV), Equine infectious anaemia virus (EIAV) and Eastern equine encephalomyelitis virus (EEEV) were acquired by molecular cloning, and spotted on the diagnostic gene chip. The cDNAs reverse-transcribed from RNAs of samples were labelled with Cy5-dUTP/Cy3-dUTP as fluorescent probes. Following specific hybridization of the deposited gene chip and labelled probes, fluorescence signals were scanned by laser scanner and the resulting image was analysed by QiamtArray software on a digital computer. The results showed that the prepared gene chip could detect and distinguish the five equine viruses. Its sensitivity was about 25 viral genome copies. The hybridization specificity was confirmed by the presence of red fluorescence signals on the corresponding sites with samples from the five relevant viruses in horses and by the absence of positive signals with the specimens from irrelevant viruses from other animals. Peripheral blood leucocytes (PBL) from some seropositive horses in post-arrival quarantine were negative according to virus isolation, but were positive for EHV-1 and EAV according to the gene chip technique. The overall results suggest that gene chips, which are quick, specific, sensitive and reliable, can provide a practical alternative for screening quarantined animals, and will be able to deal with a large number of animal samples within a very short period of time.

To construct an stx2 gene mutant phage phi Min27(delta stx::cat) and to observe its infectiousness of various serotypes Escherichia coli strains.With the help of Red recombinant system, the stx2 gene of the E. coli O157:H7 Min27 strain isolated from intestinal feces of piglet with diarrhea at a swine farm of Shanghai, was replaced by the chloramphenicol acetyltrasferase (cat) gene from plasmid pLacI. Phage phi Min27(delta stx::cat) was isolated after induction of E. coli Min27(delta stx::cat) strain with mitomycin C. Twenty-one E. coli strains with various serotypes were infected with phi Min27(delta stx::cat), and plaque formation and lysogenic conversion of them were investigated.Of the 21 E. coli isolates, 2 with the serotypes of O60 and O138 integrated the phi Min27(delta stx::cat) in their chromosomes and expressed resistance to chloramphenicol. With the exception of one laboratory E. coli strain MG1655, none of the tested E. coli strains supported the formation of plaques and lysogenization when used as indicators for phi Min27(delta stx::cat). Following induction with mitomycin C, these lysogenic strains released infectious particles of phi Min27(delta stx::cat) that formed plaques on a lawn of E.coli laboratory strain MC1061.These results demonstrated that phi Min27(delta stx::cat) was able to infect and lysogenize particular E. coli strains and that the lysogens could produce infectious phage progeny. It could be inferred that Stx bacteriophages were able to spread exogenous genes among E. coli strains. The work provided a basis for further study on mechanisms of Stx phages infection and control of Stx expression.

Streptococcus equi ssp. zooepidemicus (SEZ) causes serious disease in pigs. M-like protein (SzP) of SEZ is a virulence factor with opsonin function. To fabricate better vaccine against SEZ, We have generated a szp -knockout strain by homologous recombination. Two fragments, upstream and downstream of the szp gene, were obtained by genome walking. The vector was then used to delete a 1090 bp segment of szp gene from a strain of SEZ (ATCC35246). The mutant strain had 1000-fold decrease in LD50 as compared with wild type, and provided 85% protection against challenge with a virulent strain when administered as a live vaccine. Semi-quantitative RT-PCR analysis showed a marked increased in levels of IL-4 and IFN-γ mRNA in immunized mice. These data demonstrate that szp -knockout strain is a better candidate for vaccine development.

The DNA encoding NS1 protein of H9N2 avian influenza virus (AIV) was prepared by RT-PCR and inserted into pET-32a(+) to construct the expression plasmid pET-NSl. The recombinant plasmid was transformed into E.coli BL21 and expressed by IPTG induction at an optimized temperature. The recombinant NS1 protein was analyzed by SDS-PAGE and Western blot. The purified NS1 protein was plated as coating antigen to develop indirect ELISA for the detection of antibody against NS1. AIV negative sera and sera collected from H9N2 AIV infected birds and vaccinated birds were tested to determine the cut-off value between vaccinated poultry and virus infected poultry, and the minimum samples statistically. The results showed that a 46 KDa recombinant NS1 protein was expressed and the optimal coating concentration was 40 mug/mL, the optimal serum dilution was 1:200, the optimal HRP-IgG dilution was 1:10000 and the optimal reaction time for antigen and antibody was lh. The cut-off value was determined 0.5. To determine the antibody level against NS1 in a flock of poultry, at least 30 sera samples should be tested. The indirect-ELISA can be applied to differential diagnosis of vaccinated or infected poultry by AIV H9N2 in group.

The general distribution of monogenean parasites of aquatic vertebrates inhabiting Chinese inland waters is summarised. Five hundred and seventy-two out of a total of 581 species of monogeneans were discovered on fish, while only nine species were found on Amphibia and Reptilia. Most dactylogyrids and diplozoids parasitise cyprinids, while ancyrocephalids occur on silurids and cyprinids, and gyrodactylids are found mainly on cyprinids and cobitids. Analyses of host-specificity and host-diversity suggests that the family Ancyrocephalidae should be divided into several families parallel with the Dactylogyridae. Of 12 subfamilies of the Cyprinidae, only the Gobiobotinae was found to be free of infection with monogeneans, and the genus Dactylogyrus has more species than any other monogenean genus associated with every cyprinid subfamily.